Production of sesterterpene ophiobolin by a bifunctional terpene synthase in Escherichia coli

Abstract

Ophiobolins (ophs) are characteristic 5-8-5 tricyclic sesterterpenes with potential pharmaceutical activities. Ophiobolin synthase is a bifunctional terpene synthase (BTS) that catalyzes both chain elongation and cyclization. In Aspergillus ustus 094102, ophiobolin accumulation was involved with not only ophiobolin synthase C25 (Au8003) but also other four gene clusters containing C15 (Au6298), C20 (Au13192 and Au11565), and C30 (Au3446) terpene synthases. In this report, overexpression of codon-optimized gene Au8003 resulted in a detectable production of oph F in E. coli. In subsequent modulation of culture conditions, pentose arabinose allowed a more than 10-fold improvement of production than that of glycerol. To achieve a higher titer, the whole mevalonate pathway and an additional copy of isopentenyl diphosphate isomerase gene were assembled, leading to approximately 24-fold and 60-fold yield increases, respectively. The above four terpene synthase genes related to ophiobolin production in strain 094102 were individually or combinatorially overexpressed with Au8003 to mimic the original fungal biosynthesis. The biosynthesis of oph scaffold was increased by short-chain terpene synthases (C15 and C20), among which the C15 synthase gene contributed the highest yield of 82.76 mg/L at 96 h; the multi-gene combinatorial results suggested that cyclization might be a rate-limiting step. Further protein engineering including fusion tags and phylogenetically based mutations on the rate-limiting cyclization part of Au8003 enabled a further yield improvement (> 150 mg/L at 96 h) in shake flasks. These multiple approaches for sesterterpene skeleton production using engineered E. coli may be applicable for cost-effective, high-yield productions of ophiobolins and other compounds synthesized by BTSs.